Arsenazo III has been used to measure free calcium in cells and tissues; however, its binding characteristics with calcium are not well understood. The objective of this investigation was to accurately determine the arsenazo-calcium stoichiometry so that it could be used to measure free calcium in cells and membranes. At low arsenazo III concentrations (9 Mum), the stoichiometry of arsenazo-calcium interactions was found to be pH dependent, even in the narrow range of pH 6.55 to 7.32. The binding stoichiometry was 1:1 in the absence of EGTA, while it varied with pH in the presence of EGTA. At lower pH (6.55-7.0) it was 1:1 and it gradually changed to 2:1 (calcium:arsenazo III) at higher pH (7.32). At higher arsenazo III concentration (100 Mum) the stoichiometry was 1:1 for the pH range 6.55 to 7.32.